Analysis of the Structural Elements Required for Highly Efficient Catalysis

黄毒Trimeresurus flavoviridis毒液中一个活性较高的asp -49-磷脂酶A_2 （PLA_2）和两个活性仅为Asp-49-PLA_2的1-2%的Lys-49-PLA_2含有122个氨基酸残基，它们的序列同源性为60%，14个半胱氨酸残基的位置相同。这种PLA_2同工酶为阐明激发催化功能和各种生理功能所需的结构元件提供了宝贵的系统。本研究的目的是通过合成黄病毒T. PLA_2型突变蛋白来研究PLA_2的结构-功能关系。(1)通过对硝基苯磺酸甲酯甲基化His47，研究了Asp-49-PLA_2活性位点His47- asp99偶对的ph依赖性。His47的质子化似乎稳定了短暂的催化状态。(2)制备了3个Asp-49-PLA_2 cdna和2个Lys-49-PLA_2 cdna。Asp-49-PLA_2的cDNA序列与用该蛋白测定的氨基酸序列有部分不一致。对该蛋白序列的重新研究证实了cDNA序列的正确性。并对与Asp-49-PLA_2相似的PLA_2 cDNA序列PL-X进行了测序。利用重组DNA技术表达这些蛋白尚未成功。深入研究正在进行中。(3)对编码4种不同PLA_2的4种cdna进行比较，发现5′-区和3′-非编码区核苷酸序列同源性比编码区高得多。作为寻找非编码区在调控和表达中的作用的一步，基因组DNA的分析正在进行中。九个克隆人中的一个让我们接受了分析。(4)虽然在此期间PLA_2蛋白的表达尚未成功，但发现了黄病毒T. PLA_2同工酶的独特核苷酸序列，为今后的开发奠定了基础。少

A highly reactive Asp-49-phospholipase A_2 (PLA_2) and two Lys-49-PLA_2, whose activities are only 1-2% that of Asp-49-PLA_2, from Trimeresurus flavoviridis venom contain 122 amino acid residues, and their sequences are 60% homologous and the positions of the 14 half-cystine residues are identical. Such PLA_2 isozymes provide an invaluable system for elucidating the structural elements required for eliciting catalytic function and various physiological functions. The aim of this research is to study the structure-function relationships of PLA_2 through synthesis of mutant proteins of the types of T. flavoviridis PLA_2. (1) As a step of this study, an active site His47-Asp99 couple in Asp-49-PLA_2 was investigated by pH-dependent methylation of His47 with methyl p-nitrobenzenesulfonate. Protonation of His47 appears to stabilize a transient state of catalysis. (2) Three cDNAs for Asp-49-PLA_2 and two Lys-49-PLA_2 were prepared. The amino acid sequence deduced from the uncleotide sequence … More of cDNA of Asp-49-PLA_2 was partially inconsistent with the amino acid sequence determined with the protein. The reinvestigation of the protein sequence established that the sequence from cDNA is correct. The nucleotide sequence of a cDNA encoding a PLA_2 which is similar to an Asp-49-PLA_2 called PL-X was also sequenced. Expression of the proteins using the cDNAs by recombinant DNA technology has not yet been successful. Intensive study is in progress. (3) Comparison of the four cDNAs encoding four different PLA_2 led to an interesting discovery that the nucleotide sequences of 5'- and 3'-noncoding regions are much more homologous than those of the coding regions. As a step for searching the roles of the noncoding regions in regulation and expression, analysis of genome DNA is on the way. One of nine clones obtained us under analysis. (4) Although expression of PLA_2 proteins has not yet been successful during the period, the finding of the unique nucleotide sequences for T. flavoviridis PLA_2 isozymes established the basis for future development. Less